【摘要】 为建立蓝舌病病毒(BTV)的反向遗传操作系统,本研究构建了分别表达血清1型BTV SZ97/1株VP1、VP3、VP4、VP6、VP7、NS1和NS2蛋白的7个真核表达质粒,同时又构建了含有SZ97/1株BTV的全部10个基因节段的T7聚合酶体外转录重组质粒。并利用T7 RNA聚合酶对酶切线性化的10个体外转录质粒进行体外转录,制备BTV全部10个基因的体外转录RNA。将已构建的7个真核表达质粒和体外转录获得的RNA产物依次共转染BHK-21细胞,拯救出含有分子标记的BTV。本研究首次在我国建立了BTV的反向遗传操作系统,为我国深入开展该病毒致病机理、传播机制、基因组功能的研究以及新型疫苗的研发奠定了基础。更多还原

【Abstract】 To establish a reverse genetics system of bluetongue virus(BTV),seven additional eukaryotic expression recombinant plasmids were generated which expressed seven proteins(VP1,VP3,VP4,VP6,VP7,NS1,and NS2) of serotype 1BTV strain SZ97/1,respectively.and ten T7 in vitro transcription recombinant plasmids for BTV transcripts were constructed for all 10 segments of the SZ97/1 virus genome,respectively.BTV transcripts were produced from the linearized T7 transcription recombinant plasmids using T7 RNA polymerase in vitro transcription Kit.In addition,the BTV with genetic marker of Afl Ⅱrestriction enzyme site introduced in segment 10 was rescued from BHK-21 cells,which were first co-transfected with these seven additional eukaryotic expression recombinant plasmids,and followed by second transfection with a complete set of the ten T7transcripts of BTV at 18 hours post initial transfection.In the present study,a reverse genetics system of BTV was developed which was the first to be reported the establishment of reverse genetics system for BTV in China.This technique platform would not only facilitate further investigations of the pathogenesis,mechanism of transmission,and genomeprotein function of BTV,but also provide a tool to develop novel type vaccine against bluetongue disease.更多还原